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Biomedical subjects

G Kemball-Cook

Publications and source records attributed to G Kemball-Cook.

At least 55 records · Page 3Linked to original sources
PubMed1993

The behaviour of different factor VIII concentrates in a chromogenic factor X-activating system.

A chromogenic factor Xa generation method has been developed for comparing the co-factor activity of factor VIII concentrates at physiological factor VIII concentrations (1 iu/ml). In the presence of thrombin all concentrates gave similar rapid rates of factor Xa generation, but in the absence of thrombin there were major differences between the rates of Xa generation between different products. High purity products, particularly those prepared by monoclonal antibody purification from plasma and recombinant sources, gave more rapid Xa generation than most intermediate-purity products. There was a very strong correlation between the rate of Xa generation and the difference in factor VIII potency by one-stage and two-stage assays. These results suggest the possible presence of small amounts of activated factor VIII in some concentrates, but differences in von Willebrand factor content could also contribute towards the different rates of factor Xa generation observed.

Blood Chemical Analysis↗
PubMed1992

Interaction of factor VIII with phospholipids: role of composition and negative charge.

Radiolabelled human anti-FVIII:C antibody was affinity-purified according to its ability to bind to factor VIII-phospholipid (FVIII-PL) complexes, yielding a fraction directed against the phospholipid binding-site (PL-site antibody). This antibody was used as a specific probe for FVIII binding to PL vesicles containing a variety of natural and synthetic PLs. Of purified PLs tested for FVIII binding, phosphatidyl serine (PS) and phosphatidic acid (PA) were highly active, phosphatidyl inositol (PI) much less so, and both phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) inactive: the apparent dissociation constant (Kd app) for FVIII binding to PS:PC vesicles showed a strong dependence on PS content. Free-flow electrophoresis of vesicles confirmed FVIII binding to PS:PC required both net negative charge and specific head-group: neither PS vesicles given a positive charge with stearylamine nor PC vesicles made negative with dicetyl phosphate bound FVIII. It is concluded that the negative charge required for FVIII binding must be presented on the phospholipid surface in the correct orientation: phosphatidyl serine supplies this charge in coagulant-active PL preparations.

Amines↗
PubMed1991

Proteolysis of factor VIII heavy chain polypeptides in plasma and concentrates.

Factor VIII heavy chain (FVIII HC) polypeptides have been studied in both normal plasma and FVIII concentrates on exposure to three coagulation proteases. FVIII samples were incubated with labelled affinity-purified anti-FVIII Fab' fragments, immunocomplexes formed were visualized by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and apparent relative molecular masses (Mr) of each band assigned. FVIII HC polypeptides were detected in all types of samples, including plasma, without further purification. Normal plasma contained a range of polypeptides with the largest dominant band at a net apparent Mr of 250-300 kD, and the smallest at 80-90 kD: the bands visualized correspond to the 90-210 kD HC species seen on conventional analysis of purified FVIII. No bands were produced from samples of haemophilic plasma. Treatment of plasma or FVIII concentrate with low concentrations (1 IU/ml) of thrombin removed the 250-300 kD and other intermediate bands, intensified then removed the 80-90 kD polypeptide and produced a band at 40-50 kD. Thrombin-associated rise and fall in FVIII clotting activity by one-stage assay correlated with intensity of the 80-90 kD polypeptide. A polypeptide of Mr 40-50 kD was also produced after incubation with activated factor X: activated factor VII plus thromboplastin had no effect on HC structure. FVIII polypeptides were visualized in prothrombin complex concentrates, with a more degraded profile seen in a deliberately 'activated' product.

Biological Products↗
PubMed1991

Variable inactivation of human factor VIII from different sources by human factor VIII inhibitors.

The source of human factor VIIII (FVIII) had a marked effect on the inhibitory activity of a panel of eight human FVIII inhibitors. Use of conventional FVIII concentrates gave lower inhibitor titres whereas a monoclonal antibody purified FVIII concentrate gave titres similar to or greater than those with plasma. Addition of phospholipid (PL) protected highly purified FVIII against inhibition. The content of PL-bound FVIII in concentrates may account for the observed differences.

Animals↗
PubMed1990

Factor VIII heavy chain polypeptides in plasma and concentrates.

Factor VIII polypeptides in plasma and FVIII concentrates have been analysed by an electrophoretic technique based on that of Weinstein et al (1981). Samples were complexed with radiolabelled anti-FVIII Fab', and the immunocomplexes visualized by SDS-polyacrylamide electrophoresis. The technique visualized FVIII heavy chain polypeptides in all types of samples, including plasma, without further purification. Fresh or frozen normal plasma (collected into protease inhibitors) contained a range of polypeptides with the largest dominant band at an apparent Mr of 250-300 kDa, and the smallest at 80-90 kDa: no bands were produced from samples of severe haemophilic plasma. Cryoprecipitate had a similar polypeptide distribution to normal plasma, but intermediate purity FVIII concentrates showed more degraded patterns which varied between products: the 250-300 kDa bands were reduced or absent, the 80-90 kDa bands were more pronounced than in plasma, and in one product a polypeptide was seen at approximately 40-50 kDa. In some products heat treatment for viral inactivation increased the proportion of smaller FVIII polypeptides. Highly-purified FVIII concentrate derived from plasma was also degraded relative to plasma FVIII, and two products obtained by recombinant DNA technology both showed degraded, though slightly different, profiles. The native structure of FVIII in fresh plasma appears heterogeneous with a predominance of higher Mr forms: these are degraded to a greater or lesser extent during concentrate production, dependent on the manufacturing processes used.

Blood Preservation↗
PubMed1989

Assay discrepancies with highly purified factor VIII concentrates.

We have assayed two different monoclonal-antibody-purified concentrates (A and B) and one conventional concentrate (C), against the 3rd International Standard for factor VIII concentrate, using one-stage, two-stage and chromogenic methods. One-stage assays performed with immunodepleted plasmas gave lower potencies than with haemophilic plasma for all concentrates, though the discrepancies were most marked for the two highly purified products. The absence of von Willebrand factor in one of the immunodepleted plasmas appeared to contribute towards the low potencies observed. In addition, potencies of product A were 50% higher by one-stage assays (haemophilic plasma) than by two-stage or chromogenic methods. These results indicate the need for careful evaluation of assay methodologies for assessment of factor VIII:C activity in highly purified concentrates.

Blood Proteins↗
PubMed1988

Factor VIII procoagulant protein interacts with phospholipid vesicles via its 80 kDa light chain.

In a previous report, we detailed fractionation of polyclonal human anti-Factor VIII:C into a component directed exclusively against the phospholipid-binding site on Factor VIII (PL-site antibody) and another directed at other sites (non-PL-site antibody). The location on the F.VIII molecule of its PL-binding site has now been studied by two different methods using this fractionated 125I-labelled anti-F.VIII:C Fab'. The first method was modified from that of Weinstein et al. (Proc Natl Acad Sci USA 1981; 78: 5137-41), involving electrophoresis of F.VIII peptide-125I-Fab' A/F.VIII immunocomplexes in SDS-polyacrylamide gels. PL-site antibody reacted with F.VIII peptides of apparent Mr approximately 80 kDa and sometimes 160 kDa in plasma and concentrate, but not with larger peptides. Non-PL-site antibody, however, reacted with a range of peptides of apparent Mr 90 kDa to 280 kDa. In addition, when purified F.VIII containing heavy and light chains (HC + LC), and isolated LC peptides were analysed, PL-site antibody bound to LC peptides whereas non-PL-site antibody did not. The second method used the antibody pools in immunoradiometric assays (IRMA's) of purified F.VIII peptides. Both labels measured similar amounts of F.VIII:Ag in a sample of purified F.VIII containing both HC and LC; on assaying an HC preparation, however, PL-site label measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites are absent in HC preparations. These results indicate that F.VIII binds to PL via its 80 kDa light chain.

Antigen-Antibody Complex↗
PubMed1987

The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides.

The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results.

Antithrombin III↗
PubMed1986

Factor VIII concentrates contain factor VIII procoagulant antigen bound to phospholipid.

Fractionation of a human antibody to factor VIII: Ag by immunoabsorption with factor VIII/phospholipid (PL) complex has produced two pools of labelled Fab' fragments which can be used in fluid-phase immunoradiometric assays (IRMAs). One pool binds only to the PL-binding sites on factor VIII:Ag (and thus measures only that factor VIII:Ag in a sample not bound to PL), while the second binds to other antigenic sites. Parallel assays of factor VIII-containing materials using these two pools provide estimates of the proportion of factor VIII: Ag bound to PL in those materials. Five batches each of nine brands of factor VIII concentrates from eight different manufacturers were tested for PL-bound factor VIII:Ag by this method: all contained substantial amounts, ranging from 28% to 54% of the total factor VIII:Ag present. In addition, varying amounts of the factor VIII:Ag present in both non-activated and activated prothrombin complex concentrates (PCCs) from four manufacturers were PL-bound.

Antigens↗
PubMed1984

Fractionation of human antibody to factor VIII:C: and IRMA for phospholipid binding sites on factor VIII C:Ag.

Labelled Fab' fragments, derived from the plasma of a severe haemophiliac with antibody directed against factor VIII clotting antigen (VIII C:Ag), were fractionated by immunoabsorption with, first, a complex of phospholipid (PL) vesicles and factor VIII and, second, with factor VIII alone. Two pools of labelled anti-VIII C:Ag were obtained and were used in immunoradiometric assays (IRMAs) for VIII C:Ag. With one pool (non-PL-site antibody) VIII C:Ag assays were unaffected by pre-incubation of factor VIII with PL vesicles; however, binding of the second pool of antibody to VIII C:Ag was prevented by PL preincubation, indicating that these antibody molecules bind at or near a phospholipid binding site on VIII C:Ag (PL-site antibody). Assays of VIII C:Ag in an intermediate purity factor VIII concentrate with these two antibody pools indicate that more than one third of the VIII C:Ag may be bound to PL.

Antibodies↗
PubMed1983

Binding to phospholipid protects factor VIII from inactivation by human antibodies.

The addition of purified factor IXa and phospholipid to factor VIII concentrate protected the VIII:C from inactivation by human antibodies. This protective effect was shown to be due largely to the phospholipid. Addition of phospholipid alone gave substantial protection against even high-titer antibodies, as shown by measurements of thrombin generation and VIII:C assays. Increasing concentrations of phospholipid led to significant reductions in the amount of VIII C:Ag detected by an IRMA method, up to 70% of the original VIII C:Ag being "lost" at the highest concentration of phospholipid. These results indicate that phospholipid binding plays an important part in the procoagulant activity of factor VIII and that human antibodies to VIII:C are directed largely at the phospholipid binding site. The addition of phospholipid to factor VIII concentrates could have important clinical applications in the treatment of hemophiliacs with antibodies to factor VIII.

Antibodies↗
PubMed1981

Factor VIII-related activities in therapeutic concentrates.

Recent studies have suggested that factor VIII exists in plasma as a complex containing two distinct antigens (VIII R:Ag and VIII C:Ag) and two different biological activities (VIII:C and VIII R:RCo). Concentrations of these four entities have been measured in all the different types of therapeutic material used for treatment of hemophilia and von Willebrand's disease. In all materials, the ratio of VIII R:Ag to VIII:C was greater than 1: however, there were significant differences between concentrates, and the lowest ratio was found in cryoprecipitate. In VIII R:Ag assays, the freeze-dried concentrates gave nonparallel dose-response curves compared with plasma, indicating alterations in molecular form during purification. Values for VIII C:Ag were much lower than the corresponding VIII R:Ag values, and ratios of VIII C:Ag to VIII:C were close to 1 in many of the concentrates, suggesting that the low yields of VIII:C in some production processes could result from losses of VIII:C molecules rather than from denaturation. Measurement of VIII R:RCo by a platelet counting method gave lower values in most concentrates than by an aggregometry technique. It is proposed that the platelet counting method may reflect closely the biological activity of the concentrates in treatment of von Willebrand's disease.

Antigens↗
PubMed1979

Anticoagulant activities of high and low molecular weight heparin fractions.

The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti-Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti-Xa assays, but in APTT assays using synthetic substrate S-2222 gave about 20% lower levels than anti-Xa clotting assays for both heparins. Complete protamine neutralization of the post-injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti-Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three assays in vitro. This non-neutralizable activity probably accounts for the difference between the anti-Xa clotting and synthetic substrate assays. Studies by crossed immunoelectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.

Adult↗